Antibody System Anti-Insulin Glargine Neutralizing Antibody ELISA Kit
Overview
This enzyme-linked immunosorbent assay (ELISA) kit is engineered for the precise quantitative determination of neutralizing antibodies against Insulin Glargine in serum and plasma samples. Insulin Glargine is a long-acting, recombinant human insulin analog modified with a glycine substitution at position A21 and two arginine residues added to the C-terminus of the B-chain. These structural alterations shift its isoelectric point, causing it to form micro-precipitates at physiological subcutaneous pH that slowly release insulin monomers to provide a flat, peakless basal glycemic profile over 24 hours. While clinically effective for managing Type 1 and Type 2 diabetes mellitus, chronic administration can provoke an immune response leading to the generation of anti-drug antibodies (ADAs). Neutralizing antibodies are a critical subset of ADAs that explicitly bind to the receptor-interaction domains of the analog, physically blocking it from engaging the endogenous human insulin receptor. This inhibition compromises downstream tyrosine kinase cell signaling cascades, driving therapeutic resistance and loss of glycemic control. This competitive ELISA serves as a vital analytical instrument for clinical investigators and biomanufacturers profiling long-term immunogenicity, monitoring therapeutic efficacy, and assessing bioequivalence in insulin biosimilar discovery pipelines.
PRODUCT INFO
Principle of the Assay
This assay utilizes a high-performance, solid-phase quantitative competitive enzyme immunoassay technique to measure neutralizing anti-drug antibody levels:
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Target Capture: Recombinant Insulin Glargine is uniformly pre-coated onto the solid-phase surface of a 96-well microplate.
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Competitive Binding: Calibrated reference standards or unknown biological samples are premixed with a biotin-labeled human insulin receptor tracer and pipetted directly into the microwells. Endogenous Anti-Insulin Glargine Neutralizing Antibodies present within the sample compete directly with the biotinylated receptor tracer for a limited number of immobilized Insulin Glargine binding configurations.
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Enzyme Conjugation: Unbound matrix elements and non-neutralizing fractions are cleared via a rigorous wash cycle. Streptavidin conjugated to Horseradish Peroxidase (Streptavidin-HRP) is introduced into the wells, anchoring specifically to the remaining captured biotin-labeled receptor domains.
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Signal Amplification and Readout: Following a final wash to remove excess enzyme reagent, a colorimetric substrate solution is added to initiate enzymatic signal development. Because this is a competitive assay format where neutralizing antibodies physically prevent receptor tracer binding, the resulting color intensity develops in inverse proportion to the concentration of active Anti-Insulin Glargine Neutralizing Antibodies bound in the initial step. The reaction is terminated with a stop solution, and absorbance is quantified spectrophotometrically.
Key Features and Performance Metrics
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Refined Detection Capability: Exhibits an analytical sensitivity down to 98.78 ng/mL, allowing investigators to track low-affinity or early-stage neutralizing antibody formations.
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Extended Quantitative Range: Delivers a reliable linear tracking range from 156.25 ng/mL to 10,000 ng/mL to seamlessly accommodate diverse sample concentration profiles without immediate saturation.
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Consistent Intra-Assay Precision: Demonstrates reliable target reproducibility with a Coefficient of Variation (CV) of less than 20 percent, validated through sixteen separate replicate evaluations across three samples of known concentration on a single test plate.
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Outstanding Inter-Assay Uniformity: Confirms high day-to-day and batch-to-batch experimental reliability with a CV of less than 20 percent, verified across twenty-four distinct assay runs using three independent reference concentration samples.
Technical Specifications
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Catalog No.: KAB94003
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Assay Configuration: Quantitative Competitive Enzyme Immunoassay
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Targeted Biomarker: Anti-Insulin Glargine Neutralizing Antibodies
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Validated Matrices: Serum and Plasma
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Assay Range: 156.25 to 10,000 ng/mL
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Analytical Sensitivity: 98.78 ng/mL
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Solid-Phase Target: Recombinant Insulin Glargine
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Detection System: Biotin-Insulin Receptor / Streptavidin-HRP Conjugate
Applications and Workflow Summary
This competitive ELISA platform is highly optimized for therapeutic drug immunogenicity monitoring, biosimilar validation, and downstream pharmacokinetic profiling. Investigators should observe the following guidelines:
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Dilution and Layout: Unknown serum or plasma matrices must be screened alongside the provided reference standards. Samples exhibiting target concentrations exceeding the maximum range of 10,000 ng/mL must be diluted empirically in standard assay matrix buffer prior to running the assay.
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Analysis Requirements: To achieve absolute quantification accuracy, investigators must generate a standard curve simultaneously during every plate run, plotting standard absorbance profiles against log-transformed concentration values.
Handling, Stability and Storage
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Kit Shipping: Distributed under secure, temperature-controlled conditions on blue ice or chilled gel packs to preserve protein matrix and enzyme binding configurations.
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Operational Stability: Built with high-grade components to guarantee consistent, low-decay signal retention over a standard six-month framework when kept at the manufacturer-specified storage parameters.
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Storage Guidance: Keep the complete kit stored strictly at the recommended refrigeration temperature upon receipt. Ensure that unused microplate wells remain sealed within their original desiccant pouch to guard against moisture compromise.
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